We will continue our studies of specificity in protein/DNA interactions. Two proteins and their binding sites will be studied in detail: 1. The Mnt repressor protein of bacteriophage P22 will be analyzed for its quantitative specificity, the change in affinity that results from all possible changes in the operator sequence. This study will be enhanced by the employment of a 'quantitative sequencing' protocol that allows one to sensitively measure the relative proportions of each sequence in a mixture of different sequences. We will also examine the non-independent interactions of the protein with different positions of the operator using both directed and randomized strategies. We will further examine changes in specificity of mutant Mnt proteins, including measurements of the quantitative specificity of particular mutations in the DNA contact region of the protein. We will also select randomized proteins which can recognize particular operator sequences. These methods will provide a large database of amino acid/nucleic acid contacts that show specific binding. 2, The T7RNA polymerase interaction with its promoter sites will be studied using quantitative in vivo assays. The activities of a collection of varied promoters will be analyzed for the effects of each base at each promoter position and also for non-independent interactions between promoter positions. We will also examine how the sequence changes affect particular kinetic steps of transcription initiation, both from the in vivo measurements of promoter activity and from in vitro measurements of those kinetic parameters.